The structural, functional and sheer mechanistic complexity of the neurotransmitter transporters (NTs) remains a significant challenge that we must continue to address because these cell components are responsible, as a class - the Neurotransmitter : Sodium Symporter (NSS) - and individually (DAT, SERT, NET) for major aspects of neuronal function in the brain. These membrane proteins play essential roles in cell signaling and, not surprisingly, are primary targets for medications (e.g., the antidepressants) as well as for abused drugs including the psychostimulants cocaine and amphetamine. The organization of the PPG and its Research Plan, in the 3 individual Projects and the 2 Cores, recognizes the rapid and abundant new developments in the field and new opportunities to understand the mechanisms of the NTs in the physiological processes underlying neurotransmission in general, and their involvement in drug addiction and abuse in particular. The long-term goals are to understand how the accumulation and efflux of neurotransmitters (e.g., dopamine) are regulated by post-translational modification of the NTs (DAT), by their association with other proteins, and by their localization to specific membrane micro-domains. Mechanistic details are sought to understand the molecular and cellular processes governing the activity and availability of the NTs, such as DAT, in the presynaptic membrane, and how these processes are altered in diseased states involving signaling dysfunction. With appreciation for the multidisciplinary, multiscale strategies required to achieve such goals, this renewal application builds upon the collaborative acumen and synergies established in the PPG consortium, and widens the focus to include elucidation of functional mechanisms in large membrane assemblies (Project 1), whole cells, tissues, and even whole animals (Projects 2&3 and Core B). To address these mechanisms in DAT and SERT, our research plan leverages as well new methodology, major equipment and expertise in many cognate areas, through extensive collaborations that the participating labs have established to create a multidisciplinary team even larger than the PPG, which that enhances and widens the power and scope of the innovative research proposed in this application. PUBLIC HEALTH RELEVANCE: The dopamine transporter is responsible for the rewarding properties and abuse potential of cocaine, amphetamine, and related psychostimulants. The related transporters for serotonin and norepinephrine are targets for antidepressant medications and secondary targets for psychostimulants. We strive to understand how these transporters are regulated in health and in disease. OVERALL PROJECT